The lethal mechanism of lymphocyte-mediated cytotoxicity of tumor cells is unknown. Specific irreversible inhibitors, e.g., alpha-1-antichymotrypsin and haptenic active site reactants, were instrumental to our initial, indirect approaches which indicate that a serine proteinase with aromatic amino acid specificity must have a crucial role in human natural killer (NK) lymphocyte-mediated cytotoxicity. We will use these inhibitors to tag NK-associated proteinases for subsequent antibody affinity isolation. The molecular weights, subunit structure, pI and cellular localization of the isolated NK-associated proteinase(s) will be determined. NK cell lines and clones have been established. These and additional NK clones will be maintained as the source of materials for these studies. Major emphasis will be placed on obtaining IL-2 independent killer clones. This will be done by creating hybridomas with HAT sensitive cell lines, by transformation and by mutation. The NK proteinase may function in NK-associated degranulation, in activation of a lethal substance, or in yet unrecognized processes. We will assess the importance of degranulation in NK and the potential proteinase control of degranulation. In addition, we will examine whether prolethal substances are cleaved prior to transfer of lethal materials to the tumor cells destined to die. Because of their experimental accessibility, the proteinases that control cytotoxicity could be the first components of the lymphocytes' lethal weaponry to be isolated.